Capture, Concentration And Quantitation Of Abnormal Prion Protein From Biological Fluids Using Depth Filtration

ABSTRACT

Methods for producing biological solutions such as immunoglobulins and in particular anti-D immunoglobulin substantially free of abnormal prion protein resulting therefrom. Specifically provided are methods for aggregation of prions and depth filtration of the biological solution to capture and remove abnormal and if desired, normal prion protein. The prion protein may then be eluted from the depth filter and filter washes and concentrated sufficient for detection at limits currently required by available assays.

BACKGROUND OF THE INVENTION

Transmissible spongiform encephalopathies (TSEs) are a collection ofneurodegenerative diseases characterized by progressive dementia,ataxia, amyloid plaque formation and spongiform degeneration in thecentral nervous system (CNS) (Prusiner, S. B., 1993, Dev. Biol. Stand.80, 31-44). The causative agent in such diseases is now understood to beabnormal prion protein The fundamental event in TSEs such asCreutzfeldt-Jakob disease (CJD) in humans, bovine spongiformencephalopathy (BSE) is cattle and scrapie in sheep is the conversion ofthe normal cellular prion protein PrP^(C), into a pathogenic isoform,PrP^(sc). Accumulation of PrP^(sc) in the brain of prion-infectedanimals correlates with the rise in titer of infectious prions and isused as a diagnostic marker for prion diseases. In light of the threatof an interspecies transmission of BSE to humans, a large number ofdomestic animals must be tested for the presence of PrP^(sc) in thebrain or other suitable material. In the absence of covalentmodifications that would allow a distinction between PrP^(sc) andPrP^(C), PrP^(sc) is routinely detected in Proteinase K (PK)-treatedhomogenates by Western blotting or enzyme-linked immunosorbent assay(ELISA) utilizing the fact that PrP^(sc) but not PrP^(C) is partiallyprotease resistant. Notably, these currently available assays do nottake advantage of the fact that PrP^(sc) forms aggregates. It is nowbelieved that formation of detergent-resistant PrP^(sc) aggregates is ageneral biochemical property of PrP^(sc) even for rare prion strainswhere PrP^(sc) is sensitive to proteolytic digestion. This aggregationoccurs when the prions are exposed to an aggregation aid for exampleincluding a complexing agent.

The fatal human neurodegenerative disorder CJD has also been transmittediatrogenically via a number of routes suggesting the possibility thatthe causative agent might also be transmitted via blood products. Theidentification of a new form of human TSE, named “variant” CJD (vCJD),confirmation of an association with the agent of bovine spongiformencephalopathy (BSE) and evidence that the distribution of the agent ofvCJD in human tissues may differ from that of classical CJD suggests theexistence of a theoretical risk that blood or blood products maytransmit PrP^(sc) (see Turner et al., Blood Reviews 1998; 12:255-68).

A number of blood products are prepared for medical use from pooleddonations of human plasma including normal and specific immunoglobulins,coagulation factor concentrates and solutions of albumin. There iscurrently considerable concern about the possibility thatbiopharmaceutical products from human or animal sources may transmitTSEs. Human plasma proteins for parenteral administration inherentlycarry a risk for disease transmission. Current technology for plasmascreening and process steps for the removal or inactivation of viruseshas greatly improved the safety of these products, see Burnouf T, etal., Blood Reviews 2000; 14:94-110, in this regard. However, suitablescreening tests have not yet been developed for abnormal PrP^(sc), whichare also extremely resistant to chemical and physical means ofinactivation. To determine the probability of vCJD having beentransmitted to patients by products derived from this plasma, it isnecessary to determine the transmissibility of the PrP^(sc) inclinically relevant circumstances, the extent to which procedures usedfor plasma fractionation were capable of eliminating the PrP^(sc) fromplasma products, and the extent to which the agent PrP^(sc) can bedetected in the biological product using available assays.

Human plasma is obtained from whole blood following removal of thelarger cellular fractions. Recent studies performed by the plasmafractionation industry have demonstrated that process steps used in themanufacture of human plasma products may reduce PrP^(sc) (see Foster P.,Trans. Med. 1999, 9:3-14; Lee D C et al., J. Virol. Methods 2000,84:77-89; Foster P. et al., Vox Sang 2000, 78:86-95, and Lee D. et al.,Transfusion 2001, 41:449-55.) These process steps include Cohnfractionation, depth filtration and chromatography. Foster et al. (VoxSang, supra.) demonstrated that depth filtration was effective inremoving significant amounts of abnormal prion protein (PrP^(SC)) fromboth immunoglobulin and albumin.

There is therefore a need to develop methods of capture and removal ofthe abnormal infective prions from animal or human derived medicinalproducts or food products which are effective yet do not substantiallydegrade and/or remove the biological activity or food value of theproduct. Due to the limitations of the current methods of detection andquantitation of abnormal prions, there is an unmet need in ability toconcentrate to above detection limits and thereafter detect andaccurately quantitate the abnormal prion protein (PrP^(SC)) from thesample.

The instant invention is based on the surprising discovery that depthfiltration of aqueous liquids containing biological products, such asfor example a biologically active protein, with one or more depthfilters having a pore size less than six microns, is surprisinglyeffective in removing abnormal infective prion proteins. Moreparticularly, these inventors have made the surprising discovery thatdepth filtration of aqueous liquids containing biological products, suchas for example a biologically active protein, with one or more depthfilters having a pore size less than six microns, after treatment withan aggregation aid, is surprisingly effective in removing abnormalinfective prion proteins.

The invention provides a method for the capture, removal, concentrationand subsequent accurate quantitation of PrP^(sc) associated with TSEs,when such TSEs are contained in biological or food products.

In particular, the invention provides a method for said capture,removal, concentration and subsequent accurate quantitation of PrP^(sc)associated with TSEs, in biologicals that have been treated with one ormore aggregation aids which results in aggregation of the PrP^(sc) suchthat the PrP^(sc) will be captured in and on a filter. Any method thatresults in such aggregation may be employed as an aggregation aid ascontemplated herein. In particular it has been found that solvents suchas for example alcohols may be employed. In the methods of theinvention, an aggregation aid such as a solvent liquid that has beenadmixed with the biological or food product is passed through a filterformed of a matrix of cellulose fiber impregnated with diatomaceousearth or similar filter material which may be coated with a cationicresin having an average pore diameter of the filter ranging from 0.1micron to 6 micron. Typically the filter may be a single use disposablefilter.

In particular, the invention provides a method for the capture, removal,concentration and subsequent accurate quantitation of PrP^(sc)associated with TSEs in biologicals that have been treated with one ormore aggregation aids, for example solvent such as for example analcohol, such as for example alcohol-fractionated immunoglobulinsolutions, which comprises passing the solvent liquid containing thebiological or food product through a depth filter formed of a matrixcomprising solid particles of porous material and having a pore sizeproviding a retention less than 6 μm. Typically the filter will be asingle use disposable filter. The treatment with the aggregation aid(s)may be accomplished with the one or more aids admixed together or usedin series.

By the terms “removal” or “capture” is meant the actual physical removalof the PrP^(sc) from the liquid containing the desired protein. Forpractical purposes, the recovery of the desired protein in its originalbiological state should be substantially maintained at least to a levelin excess of 50%, preferably 80%, more preferably >90%.

Using the methods of the invention, removal of the abnormal infectiveprion protein may be achieved to an extent of at least 10^(2.5), 10³,preferably 10⁴, more particularly >10⁵.

Aside from removal of the infective PrP^(sc) from the biological or foodproduct, the invention also relates to the elution from the one or morefilters and subsequent concentration of the captured and eluted PrP^(sc)using an elution buffer which may comprise, for example, hypertonicsolutions such as for example high salt solutions so the PrP^(sc) may beaccurately quantitated using available assays.

Thus, the instant invention provides for aggregation of prions followedby filtration for the purification of a biological or food solution, theelution of the prions from the filter and the concentration of thePrP^(sc) so as to enable one skilled in the art to employ availableassays to quantitate both total prion and PrP^(sc) in a biological orfood sample. The invention will further allow the rapid high-throughputtesting of large numbers of samples for PrP^(sc).

The invention also relates to the treated biological or food solution.

Since the source of human plasma is whole blood following removal of thelarger cellular fractions, we therefore, in order to simulate the stateexpected of a TSE agent in plasma for fractionation, herein used as aninoculum a fraction of scrapie-infected hamster brain from which intactcells and larger fragments had been removed. TSE diseases are believedto be transmitted either by protease-K-resistant, conformationallyabnormal prion protein (PrP^(SC)). We herein disclose an in vitro methodof analysis to determine the distribution of hamster-adapted scrapiePrP^(sc) as a marker for the partitioning behavior of vCJD.

TSE agents are highly resistant to inactivation, therefore reduction ofany product-associated risk will be dependent on the physical removal ofinfective material during product manufacture. Process technologies usedin the manufacture of plasma products include the separation of proteinsby precipitation and chromatography with resultant protein solutionsbeing clarified and sterilized by depth and membrane filtrationprocedures, respectively. Some of these technologies by theirmodification with the methods of this invention, may be capable ofremoving TSE agents from a product stream.

PrP protein was detected herein using a Western Blot with the monoclonalantibody 3F4 specific for hamster PrP. This antibody reacts withresidues 109-112 PrP from only humans, hamsters and felines. Incubationwith 3F4 antibody was at a concentration of 0.6 ug/ml for a minimum of 1hour, after which excess antibody was washed away and the membranesincubated with a rabbit anti-mouse horseradish peroxidase conjugate(1:1000 dilution) for a minimum of 1 hour. After extensive washing withTTBS, the membranes were developed using enhanced chemiluminescence.

In the manufacture of RhoGAm® RHO(D) Immune Globulin (Human) by thisAssignee, PrP^(sc) was removed to the limit of detection during depthfiltration steps that are also used in the manufacture ofimmunoglobulins.

Western blotting is a method used to identify and characterize PrP^(sc).The PrP^(sc) is isolated by extraction and is differentiated by itspartial resistance to proteinase K digestion. The PrP^(RES) (PrP^(sc)resistant to proteinase digestion) is identified by the migrationpositions of the glycosylation forms and fragments. The sensitivity ofthis assay is approximately 3 logs less sensitive than the infectivityassay. This sensitivity issue is partially overcome by centrifuging theenzyme digested preparation, removing the supernatant and resuspendingthe prion material in a smaller volume, resulting in a concentration ofthe prion material. We have shown that the prions can be easilyconcentrated by filtering them through a filter after treatment with anaggregation aid, and later collected in a small volume by elution. Thistechnique can be used on a large scale to remove prions from a productstream.

This procedure will have a major impact on the use of the Western blotand indeed any other prion detection assay, to determine the presence ofPrP^(sc) in a biological matrix. This invention allows the TSE materialto be quantitatively concentrated quickly to allow for enhanceddetection. When seeking to purify a biological, food or cosmeticsolution of PrP^(sc), this invention has the advantage in the ease inwhich the biological, food or cosmetic solution filters through thelarge nominal pore size of the filter.

The methods of the invention are useful for the treatment ofbiologicals, foods and cosmetics by removing, eluting and, further,quantitating PrP^(sc), and depending on the aggregation aid(s) employed,PrP^(C). Among the biologicals that can be so treated are blood andblood components such as whole blood, blood serum and plasma, urine,cerebrospinal fluid and blood-derived biological products such asantibodies and immunoglobulins. One such antibody is the IgGimmunoglobulin known as monoclonal anti-D immunoglobulin or RhoGAM® Rho(D) Immune Globulin (Human). This polyclonal immunoglobulin is used inthe prevention of hemolytic disease of newborn wherein the mother isinjected with Rho(D) immunoglobulin of human origin. Such a product isRhoGAM®, available from the assignee hereof, and it operates bypreventing the unimmunized Rho (D) negative mother from responding toRho (D) antigen present on red cells and ‘received’ from an Rho(D)positive infant. Thus, by preventing anti-Rho (D) production by themother, the subsequent Rho (D) positive infant of this mother isprotected from hemolytic disease of the newborn. This successful productis currently produced by a Cohn alcohol fractionation type process.

RhoGAM® Rho(D) Immune Globulin (Human) was the first successfulprophylactic use of specific antibody to achieve antibody mediatedimmune suppression. RhoGAM® is an IgG immunoglobulin solution containinganti-Rho(D) at a dose of 300 micrograms of anti-D activity per dose.RhoGAM® can be given to the nonimmunized, Rho(D) negative pregnant womanat the appropriate time prevent future disease in her Rho(D) positiveoffspring. The disease is called hemolytic disease of the newborn ormore specifically, Rh-erythroblastosis fetalis.

A smaller dose of anti-Rho(D), MICRhoGAM® Rho(D) Immune Globulin (Human)Micro-Dose (50 micrograms of anti-Rho(D)) is also sold by the Assigneehereof for treatment of women who have abortions and miscarriages attwelve weeks gestation or earlier. While the full dose protects therecipient for up to 15 ml of Rho(D) positive red cells, the smaller doseprovides protection up to 2.5 ml of Rho(D) positive red cells. RhoGAM®is used as antenatal prophylaxis at 26 to 28 weeks gestation. Otherindications include threatened abortion at any stage of gestation withcontinuation of pregnancy, abortion or termination of pregnancy at orbeyond 13 weeks gestation, abdominal trauma or genetic amniocentesis,chorionic villus sampling (CVS) and percutaneous umbilical bloodsampling (PUBS).

Most immunoglobulin injectable materials approved for use by the FDA andBureau of Biologics have been produced by the alcohol fractionationprocedure developed by Dr. E. Cohn of Harvard during the 1940s anddescribed in Cohn et al., J. Am. Chem. Soc. 68, 459 (1946), incorporatedherein by reference. This procedure coupled with the careful selectionof plasma negative for hepatitis infectivity, HIV, and other blood-bornepathogens determined by the most sensitive tests available. That theproducts produced by this procedure are indeed safe can easily bedemonstrated by the millions of non-infected recipients of product. Theinventors hereof have now found that the alcohol employed in the Cohnprocess referenced hereinabove is sufficient to act as an aggregationaid in that it causes sufficient numbers of PrP^(sc) particles toaggregate, such that PrP^(sc) can be removed to the limits of detectionusing the inventive depth filtration, and eluted and concentrated to alevel sufficient for such detection.

The solvent composition employed has minimal effect on the IgG particlebut sufficiently aggregates the PrP^(sc) sufficient to enable it to beremoved to below its level of detection using available assays.

It is therefore an object of this invention to provide a method forremoval of PrP^(sc) and if desired, PrP^(C), from biological and foodsolutions using prion aggregation aids and membrane or depth filtration.Depth filtration is preferably used.

It is also an object of the invention to remove PrP^(sc) and if desired,PrP^(C), from protein-containing liquids, particularly those derivedfrom human plasma, without unacceptable effects on the nature orbiological activity of the proteins.

It is a further object of the invention to capture, concentrate anddetect to accurate quantitation, PrP^(sc) from any biological fluidusing the methods disclosed herein.

It is an object of the instant invention to provide abnormal infectiveprion-cleared, pure immunoglobulin for injection. Such a substantiallypure product is produced using the processing methods of the invention.

It is a further object of this invention to provide a manufacturableprocess for purifying immunoglobulins from abnormal infective prionwhich is reasonable in terms of temporal, square foot and protein yieldrequirements.

It is a further object of the invention to provide a depth filter whichcan be a single use filter and may be disposed of having removedPrP^(sc) from the process stream.

It is a further object of this invention to provide a concentratedPrP^(sc) solution, by elution of said prions from the depth filter andfilter washes.

It is yet a further object of this invention to provide a rapid assayfor the assessment of PrP^(sc) in various biological materials includingbiological fluids and human blood and plasma-derived products. Use ofsuch assays as, for example, the Western Blot, require sufficient levelsof prions unavailable in non-prion-aggregated, non-filtered biologicalsolutions. This method provides a practical method to capture, elute andconcentrate prions so that they can be detected using currentlyavailable assays. Use of these novel capture and elution methodsincreases sensitivity about 3 logs, enabling reduction in the volumesneeded to perform the detection assays.

SUMMARY OF THE INVENTION

The methods of this invention are used to produce immunoglobulin(preferably monoclonal) substantially purified of abnormal prionprotein. The substantially purified immunoglobulin is for examplemonoclonal or polyclonal anti-D immunoglobulin, for example RhoGAM® orMICRhoGAM®. This immunoglobulin formulation comprises from about 4.0 to6.0% immunoglobulin by weight, and from about 80 to 200 ppm polysorbate80, more preferably about 5.0% immunoglobulin by weight, and about 130ppm polysorbate 80.

The above referenced immunoglobulin formulation is made generally by thesteps of fractionating human plasma using an aggregation aid such as forinstance an alcohol, wherein the fractionation comprises a filtrationstep; resuspending the resulting Precipitate II; admixing theresuspended Precipitate II with a high ionic strength buffer containingan excipient; and performing nanofiltration on the immunoglobulin.

The alcohol is preferably methanol and the filtration step is performedon Supernatant III in the fractionation process, using a depth filterfor instance a Cuno Zeta Plus 90S depth filter.

The methods disclose a process for the manufacture of anti-D antibodysubstantially purified of abnormal prion protein, includingfractionating human plasma in the presence of an aggregation aid such asfor instance an alcohol wherein the fractionation comprises a filtrationstep. The filtration step may employ a depth filter such as for instancea Cuno Zeta Plus 90S depth filter, having a pore size rating of fromabout 0.6 to 6 micron. The resultant supernatant, referred to in theprocess as “Supernatant III” is processed to form a precipitate (calledin the method “Precipitate II”), which is then resuspended and admixedwith processing aids and nanofiltration on the resulting anti-D antibodyperformed thereon. The processing aids may include a high ionic strengthbuffer and a non-ionic excipient, for example 150 mM NaCl-Glycine bufferand polysorbate 80.

Further disclosed herein is a process for the manufacture of biologicalproduct substantially purified of abnormal prion protein by admixing thebiological product with an aggregation aid such as a solvent sufficientto form aggregated abnormal and normal prion protein; and filtering thethusly acquired admixture with a depth filter. The biological product isblood or blood product, cerebrospinal fluid, or urine. When the productis blood, the blood may first be clinically centrifuged and the redblood cells and platelets removed from the blood prior to admixing withthe aggregation aid. After the filtering step, the red blood cells andplatelets may be added back to the blood. The depth filter may includefor example a Cuno Zeta Plus 90S depth filter. The aggregation aid maybe a solvent such as for instance an alcohol, for instance ethanol ormethanol at a concentration of from about 2% to about 100%.

Yet further disclosed herein is a method for quantitating abnormal prionprotein in a biological solution. This method may comprise admixing thebiological solution with an aggregation aid(s) such as a solventsufficient to aggregate the abnormal prion protein, filtering theadmixture with a depth filter, eluting the abnormal prion protein offthe depth filter by washing the filter with an elution buffer,optionally concentrating the elution buffer by such method ascentrifugation, and performing an assay for abnormal prion protein onthe elution buffer. The biological solution may be blood or a bloodproduct (for example an immunoglobulin), cerebrospinal fluid, or urine.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a flow sheet showing the process of fractionation of humanplasma to obtain anti-Rh globulin. During this fractionation process thematerial may be filtered to capture prion protein.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention employs one or more aggregation aids to aggregateprion in a fluid, such that prion (normal and/or abnormal) may beeluted, captured, concentrated and detected. One class of aggregationaids will aggregate both abnormal infective prion (PrP^(SC)) as well asnormal prions in a fluid, such as a biological fluid, which prionaggregates may then be removed, eluted, concentrated and eitherabnormal, normal or both types of prions accurately quantitated usingthe methods of the invention.

The invention further contemplates use of an aggregation aid which is acomplexing agent, which agent aggregates either normal prions orabnormal prions, depending on the properties of the complexing agent.Such complexing agents include metal ions such as for example Cu2+, Ni,Zn, and Ag.

The invention allows a filter such as for example a depth filter to beused even with biological fluids comprising globular protein moleculessuch as for example an immunoglobulin or antibody, without appreciableyield loss and no significant change in immunoglobulin subclass,immunoglobulin aggregate level or immunoglobulin stability.

The methods of the invention yield a biological fluid substantially freeof abnormal infective prions (PrP^(SC)) and if desired, normal prion(PrP^(C)). The methods of aggregation and filtration of the invention infact can, when aggregation aids are properly selected to do so, ensurethat all possible categories of prion, both normal and abnormal, areremoved from the product.

The process is in particular applicable to the treatment of whole blood,blood components (e.g., serum, plasma), urine, CSF, or any biologicalsuch as for example liquids containing albumin, immunoglobulins (forexample, IgG) and fragments thereof, blood coagulation factors such asFactor IX, thrombin, fibronectin, fibrinogen, Factor VIII and Factor II,VII, IX, and X and other proteins derived from plasma. It is alsoapplicable to the treatment of plasma, Factor XI, Factor XIII,hemoglobin, alpha-2-macroglobulin, haptogobin, transferrin,apolipoproteins, protein C, protein S, C-1-esterase inhibitor, enzymes(for example, streptokinase), inter-alpha-trypsin inhibitor, growthhormones and Von Willebrand factor. Naturally occurring and recombinantanalogues of the above may be treated. In addition, the invention isapplicable to the treatment of other natural products including foods,drinks, cosmetics etc. It is also applicable to other non-plasmaanimal-derived products, such as heparin and hormones.

As stated herein, the biological fluids that can be processed using themethods of the present invention include blood and blood components suchas whole blood and components thereof including blood serum and bloodplasma, urine, cerebrospinal fluid, and any biological products such asfor example antibodies and immunoglobulins. The human plasma treated andfiltered in the instant invention can be obtained by the fractionationmethods of Cohn et al. (the “Cohn process”), referenced hereinabove, bybatch or column exchange chromatography, or by affinity chromatography.In the method of producing immunoglobulin, particularly anti-Dimmunoglobulin such as RhoCAM Rho(D) Immune Globulin (Human), referenceis made herein to commonly assigned U.S. Pat. No. 6,096,872, issued Aug.1, 2000, to Van Holten et al., the contents of which are hereinincorporated by reference.

Cohn, U.S. Pat. No. 2,390,074, the contents of which are hereinincorporated by reference, discloses a method of fractionating blood bywhich gamma globulins are prepared. The gamma globulins prepared by theCohn method contain 19 S globulin, plasminogen and lipids. While thisgamma globulin is eminently suitable for prophylaxis against diseasessuch as measles and tetanus, the presence of the 19 S globulin,plasminogen and lipids are unnecessary contaminants and may decrease itseffectiveness in preventing immunization to the Rh-factor on the fetalerythrocytes.

The substantially pure anti-Rh globulin manufactured by the validatableprocesses of the present invention is prepared from human plasma whichcontains albumin, plasminogen, alpha, beta and gamma globulins andvarious lipids. Specifically, the anti-Rh globulin of the invention is agamma globulin.

The fractionation of human plasma to obtain anti-Rh globulin is carriedout according to the methods of the aforementioned Cohn et al., as wellas commonly-assigned U.S. Pat. No. 3,449,314 to Pollack et al., theteachings of which patents are hereby incorporated by reference herein.With reference to the accompanying flow sheet of FIG. 1, the ability tofractionate human plasma is dependent upon the solubility of the variouscomponents of the plasma. At each stage of the fractionation, theseparation of the fraction and the ultimate removal of those componentswhich are undesirable in the anti-Rh globulin are determined by thecritical control of pH, temperature, concentration of the precipitantand the ionic strength of the system.

Various aggregation aids may be used in the aggregation of the prionsresident in the biological fluids of the invention. There are a numberof classes of aggregation aids that can be used, all working on theprinciple of changing the characteristics (e.g., the size) of the prionwithout aggregating or otherwise adversely affecting the milieucontaining it.

It will be appreciated that some aggregation aids aggregate bothabnormal and normal prion. Aggregation aids in this class includeorganic solvents of low dielectric constant such as acetone andalcohols, which are known to precipitate proteins and have been used inthe fractionation of plasma. More particularly, the organic solventsutilized in the method of this invention include the various alcoholswhich are completely water-miscible and those that do not react withproteins, such as for example ethanol, methanol, isopropyl, isopropanol,n-propanol, isopropyl ether, ketones, aldehydes, etc., and acetone, andpreferably methanol. Other similar aggregation aids in this class thatmay be used, to the extent they are compatible with the biologicalmaterial being treated, include ammonium sulfate, caprylic acid, and thechemical agents trichloroacetic acid (TCA), dialdehydes, heteropolyacids, and lactate monohydrate C₁₈H₂₁N₃O₄H₂O.

It will further be appreciated that some aggregation aids will aggregatethe abnormal prion thereby allowing it to be removed, while leaving thenormal prion in its native state, and vice versa—this class ofaggregation aids are the complexing agents. These complexing agents bindto the prion protein and include heteropolymolybdates,heteropolytungstates, sodium phosphotungstate (NaPTA) (all of whichaggregate only abnormal prion), and the biological agents such asantibodies (monoclonal or polyclonal), the antibodies having actiondependent upon their specificities, enzymes (such as for exampleplasminogen (which aggregates only abnormal prion) and peptides,peptides having selective action dependent upon their composition. Afurther aggregation aid which is a complexing agent includes the metalion Cu2+, which aggregates normal prion. Other similar metal ions mayinclude Ni, Zn, and Ag. These agents can be employed as a prion capturemechanism when bound to a substrate. In one embodiment it iscontemplated that the complexing agents may be used in series, forinstance, the ion Cu2+ may be admixed with the biological solution andthe normal prions removed by filtration, followed by admixing theresulting biological solution filtrate with the NaPTA to complex theabnormal prions, which may then be captured, eluted and concentrated anddetected using known assay methods.

The aggregation aid methanol is preferred for prion removal fromimmunoglobulin solutions due to its comparatively lower toxicity andsafer handling e.g., explosion danger) than other organic solvents. Whensuch solvents are used they are generally present in the admixture withthe biological fluid in concentrations of about 2% to about 100% byvolume of biological. The concentration of the solvent is dictated inthe lower range by the minimum concentration required to aggregate theprions, and at the higher range by the integrity of the biological andthe filter media.

It has now been found by these inventors that the inventive processingwith aggregation aids such as those named hereinabove results in theaggregation of the prion (either or both PrP^(sc) and PrP^(C), dependingon the aggregation aids and methods used in employing them) protein, andthat using the methods of this invention, such aggregates can be removedusing filtration. Then, using the inventive methods of filtration andlater elution from the filter and the filter washes, and if desired,concentrating the eluate, the PrP^(sc) can be obtained in concentrationssufficient to enable accurate quantitation. Using the methods of theinvention it has been found that such treatment is sufficient to removethe PrP^(sc) from the immunoglobulin formulation to below its detectionlimits. The sensitivity of assays used to detect such prion is increasedby approximately 3 logs, enabling the reduction in volume and thereforeincreasing prion concentration in the sample.

For the PrP^(sc) aggregation aspect of the invention, the aggregationaids that may be used are any that are found to precipitate abnormalinfective prion protein fibrils while being compatible with thebiological materials being treated, and compatible with the filter beingused.

The filtration aspect of the invention may be carried out at anytemperature that is appropriate to the biological materials beingfiltered, indeed the conditions for filtration are mandated by thebiological, food or cosmetic product under filtration and not theconditions required to capture the prion material. In order to preventdenaturation of proteins during fractionation and filtration, thefractionation, where employed, and filtration may be preferably carriedout at low temperatures. Since protein solubility is temperaturedependent, the temperature chosen for each step of the fractionationmust be the lowest possible which permits the desired separation and/orfiltration in order to prevent denaturation. The pH conditions should bemandated by the liability of the biological product being purified.

The depth filters that may be used in the practice of the instantinvention are those depth filters that are either charged or uncharged.Examples of depth filters that may be utilized include Celite (WorldMinerals, Lompoc, Calif.), Millipore filters 75DE and SA (MilliporeCorporation, Bedford, Mass.), and Cuno 35P and Cuno Zeta Plus 90SP (CunoCorporation, Meriden, Conn.).

Most preferable for use in the instant invention are the 47 mm Cuno ZetaPlus 90SP depth filter, along with the appropriate stainless steelfilter housing, for small scale filtration work, and the 16 square footcartridges used in manufacturing processes. Preferably the filter foruse in the practice of the instant invention is a depth filter, howevernon-depth filters may be employed for removal of aggregated PrP^(sc) aslong as the filtration through these filters does not result in theclogging of said filters. Such alternate filters are for examplemembrane filters, charged or uncharged. Such filters include forexample, the disposable syringe filters Swinex or Millex 25 mm PVDFsyringe-driven filter units, 0.22 micron Opticap and Optiseal cartridges(Millipore Corporation, Bedford Mass.).

The pore size of the filters used in the practice of the removal andcapture of PrP^(sc) (and where desired, PrP^(C) aggregates of theinvention is relatively unimportant, however, the pore size can affectthe recovery of the biological product being filtered. The pore size ofthe filter matrix is preferably in the range of. 0.2 to 6 microns,particularly 0.6 to 1.5 microns. The pore size is defined in terms ofthe particle size of particles retained thereon. Typically particles ofdefined size such as dextrans or microorganisms are used for calibrationpurposes.

The pore size of the filtering units employed in the production ofsubstantially pure, abnormal infective prion-free immunoglobulinproducts of the instant invention is less than about 6 μm, mostpreferably less than about 0.6 μm. However, any filter having a cutoffrating sufficient to reduce or eliminate abnormal infective prion from aproteinaceous solution can be employed in the processing methods of theinvention. For example, for depth filters, Cuno Zeta Plus 90S filterpads (Cuno Corporation, Meriden, Conn.) may be employed, such unithaving a molecular weight pore size rating of 0.1 to 5 micron,

Similarly, filter composition should have little effect on the abilityof the filters to capture the aggregated PrP^(sc), however, recovery ofthe prion material from said filters may require elution buffers such ashigh salt solutions or surfactants.

The filter material may comprise a depth filter which generallycomprises a self binding matrix of cellulose, together with a solidporous particulate material such as Kieselguhr, perlite or diatomaceousearth.

The depth filter generally has a thickness in the range 1-10 mm,particularly 2-5 mm. The material used for the depth filter should havelittle or no effect in the desirable protein concerned. Acceptable depthfilters include the Seitz KS80 filter of pore size 0.6 to 1.5 μm, theSeitz K200P, the Cuno Delipid Del 1 mini cartridge, effective filtrationarea 27 cm³, Millipore filters, particularly the Millipore CP20, alsoincluding conventional ultrafilters such as Millipore PTHKpolyethersulfone membrane, AMICON ym-100 regenerated celluloseultrafiltration membrane, (Millipore Corporation, Bedford Mass.), PALLFiltron Omega VR, Pall Ultrapore VFDV50 (Pall Corporation, East Hills,N.Y.), along with other cartridge filters, such as the Asahi Planovaregenerated cellulose cartridge filters, (Asahi, Tokyo Japan). Otherfilters that can be used are charged depth filters such as those of E.Begerow GmbH & Co., Langenlonsheim, Germany. However, the mostpreferable embodiments herein employ the Cuno Zeta Plus 90S filter pads,47 mm filter.

The flow rate of the biological material through the filters are thoserates suitable for ensuring proper filtration of the biological materialwhile not compromising the integrity of the filter or, in the case ofthe biological material comprising large globular proteins, a rate thatdoes not compromise the structure of the proteins so as to make thepreparation unacceptable for its intended purpose. In the depthfiltration of an immunoglobulin product for example, filtration ratesrange from about 0.01 to about 20 ml/minute, more preferably about 10ml/minute, more preferably about 1 ml/minute.

The method may be carried out in the pH range of 4-10, preferably 5-9,more preferably 6-8. However, the pH range will be determined by that pHrequired to preserve the integrity of the biological being treated andthe filter employed, and not by any limitation on the aggregation orfiltration process itself.

The application of heat is unnecessary and the process can be carriedout at substantially room temperature or below, in particular at thetemperatures of −5 to +20° C., as suitable for maintaining the integrityof the biological and the filtering medium.

As stated hereinabove, an aspect of the instant invention is thetreatment of the biological fluid with an aggregation aid such as forexample a solvent sufficient to aggregate the prion contained therein soit may be captured by filtration and eluted in a concentrationsufficient for detection using known methods such as for example,Western Blot. Some biological fluids will be so treated as a function oftheir production, for example, immunoglobulins which are treated withalcohol in the Cohn process. Where the biological fluid is not alreadyso treated, it will be treated with a suitable aggregation aid such asthose stated hereinabove so as to aggregate the prions containedtherein. Such biological fluids are enumerated hereinabove and mayinclude blood and components thereof, urine and cerebrospinal fluid, aswell as immunoglobulins.

Following treatment of the biological with the aggregation aid, thefilter pad is removed from its housing and prion eluted therefrom,concentrated if desired using a process such as centrifugation, andquantitated using available assays, all in accordance with remainingaspects of the invention, all herein described.

A protease resistant prion protein isoform is present in urine ofanimals and humans affected with prion disease. Shaked et al. (2001, J.Biol. Chem. 276 (34):31479-31482) discuss steps to isolate prions fromurine. The process described in Shaked et al. requires 2-15 ml of aurine sample to be sedimented for 5 minutes at 3000 rpm and thendialyzed overnight in cellulose membrane tubes. Subsequently the urinesamples were centrifuged at high speed (100,000×g) for 1 hour at 4° C.

The Shaked et al. procedure is time consuming and is limited by theamount of sample that can easily be concentrated. Disclosed herein is aprocedure for accurately quantitating prions from a biological fluidsuch as urine, that may be accomplished in minutes and is not limited byvolume. During prion filtration and elution from urine with the methodsof the invention, up to 1 liter of urine volume may be filtered with a47 mm Cuno filter. This volume difference allows for a magnitudeincrease in the concentrating capacity of the instant procedure comparedto the current state of the art. See Example 9 herein.

When whole blood is used as the biological fluid in the invention, aquantity of it, for example 1 liter, may first be centrifuged underconditions suitable for separating the cellular component. The resultingplasma is admixed with an aggregation aid such as for example a quantityof methanol, for instance in a ratio of about 5 parts plasma to about 1part methanol, to aggregate the prion material. The admixture is gentlymixed on a rotary shaker for a period of time sufficient to aggregatethe prions present, for example for about one (1) minute. The admixtureis then passed through a filter for example a 47 mm Cuno Zeta-Plus 90Sfilter The material may then be eluted from the filter using an elutionbuffer as described herein, and the prions quantitated by any suitableassay, such as for example a Western Blot assay. If desired, thedetection limit may be further improved by including a PrP^(sc)sedimentation step. The samples were diluted and treated withProteinase-K (PK) followed by AEBSF (4-(2-aminoethyl) benzensulfonylfluoride) to inhibit proteinase activity. Following the PK treatment thesample is centrifuged at 20,000×g for 1 hour at 4° C. The pellet is thenprepared for SDS Page.

When the biological material to be treated with prion aggregation aidsis an immunoglobulin, the biological material will be so treated duringthe plasma fractionation process. With reference to FIG. 1 and Example 1herein, plasma units are pooled and then under specified conditions arecentrifuged and relevant portions are retained for further processingwith the aggregation aid, in this case, preferably methanol. At thepoint of the fractionation wherein Supernatant III is obtained, theSupernatant III fraction is filtered using a membrane or depth filter,which filtration removes the aggregated prions that may have beencontained therein. The aggregated prions captured thereby may be elutedfrom the filter and detected and quantitated using known assays.

The capture and elution procedures of the invention result in anincrease in the detection limit of the assay by greater than 100 fold,now approaching the infectivity assay detection limit. Using methodscurrently available in the art, the infectivity assay can take months toyield results, dependent upon the species under study compared to hoursfor producing results using the methods of the invention.

Following aggregation of prions resident in biological fluid, whether bysolvent or otherwise, the next steps are the prion (for example,PrP^(SC)) elution and recovery. In these steps the filter or filterpad(s) is/are removed and washed with elution buffer. One method is theplacement of the pad(s) in a receptacle such as for example a petridish, a beaker or similar suitable container, a suitable volume forexample about 15 ml to about 100 ml of elution buffer added thereto, andthe container placed on a rotary shaker at room temperature for about 25minutes. The filter-bound PrP^(sc) is thereby eluted therefrom viagentle washing with the elution buffer. Suitable elution buffers includeany aqueous buffers, such as for example, hypertonic salt solutions suchas for example 1.0-2.0M NaCl buffers, sodium acetate-methanol buffers atconcentrations of 1.0M to about 2.0M.

If desired, the aggregated prions may be further concentrated bycentrifugation or any procedure known in the art for achieving anincreased concentration.

Given the theoretical possibility for prion contamination of bloodproducts, it was especially important to elucidate the effectiveness ofdepth filtration and the mechanism for prion removal from anintermediate from immunoglobulin production RhoGAM® Ultra-FilteredRho(D) Immune Globulin (Human). In accomplishing this goal, theseinventors used scrapie brain homogenate (SBH) from scrapie-infectedhamsters, as the source of the PrP^(sc). In order to carry out suchstudies, the PrP^(sc) “spike” was first treated with detergent tosolubilize it, and sonicated to disrupt the fibrils. The spike wastreated so as to make the PrP^(sc) as small as possible so as tochallenge the filtering system. The sonicated SBH was then sequentially0.45, 0.22 and 0.1 micron membrane filtered to better define the size ofthe PrP^(sc) spike prior to spiking. A previous study (Van Holten R, etal., Transfusion (submitted for publication)) had demonstrated that thistreatment did not adversely effect the PrP^(sc) and would additionallyinsure that the particles the depth filtration would remove would becloser in size to the individual fibrils associated with infection. Areduction in PrP^(sc) after depth filtration could indicate that prionremoval was due to the fibrils adsorbing to the positively chargedfilter media, rather than by mechanical straining. The addition of thespike into the IgG diluted in a phosphate buffer/methanol mixtureresulted in flocculation of the material which resulted in a cloudyappearance.

A Cuno Zeta Plus SP charged depth filter was used to filter the RhoGAM®Rho(D) Immune Gamma Globulin (Human) that was spiked with the SBH. Uponfiltration through a Zeta Plus SP filter the cloudiness was removed, Alayer of white precipitate was observed on the filter post filtration.Upon Western blot analysis used to detect PrP^(RES) the filter materialwas void of scrapie. With a 2.0M salt wash the prion material wasrecovered from the filter. The Western Blot results are shown herein inTable 1.

Two control runs were also performed. In the first run, the PrP^(sc)spiked immunoglobulin intermediate was first filtered through a 0.22 μmfilter to insure that the PrP^(sc) did not aggregate to larger particlesthat could be removed by the depth filter through mechanical straining.In the second run, the sonicated and filtered SBH was spiked into Trisbuffered saline (TBS) instead of the immunoglobulin intermediate,followed by the depth filtration.

The depth filter removed greater than four logs of PrP^(sc) from thefiltrate of the immunoglobulin. A significant portion of the PrP^(sc)could be recovered from the immunoglobulin filtration by elution withhigh molarity NaCl solutions. The 0.22 μm prefiltration of the spikedSupernatant III removed all detectable PrP^(sc) prior to depthfiltration. Less than one log of PrP^(sc) was removed from the buffercontrol by depth filtration. See Examples 6 and 7.

It was thus found that depth filtration removed PrP^(sc) from theimmunoglobulin by mechanical straining rather than by adsorption to thefilter matrix. The immunoglobulin preparation caused the PrP^(sc) toaggregate from particles <0.1 μm in size to particles >0.22 μm, probablyas a result of the methanol in the immunoglobulin preparation. The depthfilter failed to remove PrP^(sc) from the buffer control sample.

In Example 3 herein, membrane filtration of the sonicated SBH wasperformed prior to depth filtration of the SBH spiked Supernatant III(“SupIII”) in order to insure that the depth filter would see particlesno greater than 0.1 micron in size. This would present the greatestchallenge to the depth filter and would allow characterization of themechanism of PrP^(sc) removal. The SBH was first sonicated to break upthe PrP^(sc) aggregates and facilitate the membrane filtration. Despitethe sonication, it was necessary to serially filter the SBH throughprogressively smaller filters (0.45 and 0.22 micron) to minimizeclogging of the 0.1 micron filter.

The Cuno Zeta Plus 90SP depth filter utilizes two mechanisms forparticle removal. Particles above the nominal pore size of approximately0.1 micron are retained predominately by mechanical straining. Below 0.1micron, particles with a negative charge are retained by electrokineticadsorption to the positively charged filter media (U.S. Pat. No.4,859,340). Since particles greater than 0.1 micron had been removedfrom the SBH prior to addition to the Supernatant III and subsequentdepth filtration, it appeared that the retention of the PrP^(sc) by thedepth filter was due to increase in particle size due to exposure tomethanol. However, the charge capture mechanism of removal would be ineffect when one departs from the isoelectric point of the prion beingcaptured,

Examination of the depth filter after filtration of the SBH spikedSupIII and prior to elution with the 1.0M and 2.0M NaCl solutionsrevealed a small amount of material on the surface of the depth filter,This was believed to be a precipitate formed when the SBH was added tothe SupIII, caused by the methanol present in the SupIII. In order todetermine if this precipitate contained PrP^(sc), a second run wasperformed where the SBH spiked SupIII was first pre-filtered through a0.22 micron filter prior to depth filtration. The pre-filtration removedPrP^(sc) to undetectable levels, indicating that in the prior run thePrP^(sc) was removed by precipitation and mechanical straining, ratherthan by electrostatic adherence to the depth filter. Prusiner et al.(Biochemistry 1980; 19:4883-91) demonstrated that ethanol readilyprecipitated PrP^(sc), so it is not surprising that the presence of themethanol used in this fractionation process would have the same effect.

In order to determine whether depth filtration would remove PrP^(sc) inthe absence of a precipitating alcohol, a control run was performed (seeExample 4) where the PrP^(sc) was spiked into an aqueous buffer and thendepth filtered. The lack of removal of PrP^(sc) from the buffer controlindicated that the depth filter did not retain the protein, either bymechanical means (because the PrP^(sc) had previously passed through a0.1 micron filter) nor by electrostatic adherence.

These studies indicate that previous reports on the effectiveness ofdepth filtration to remove PrP^(sc) may be misleading. Indeed, depthfiltration does remove PrP^(sc), not by the absorptive mechanism usuallyassociated with depth filtration but by mechanical straining of theprecipitated protein. The results of this study indicate that depthfiltration alone is ineffective in removing PrP^(sc). However, when usedin conjunction with a prior precipitation step, depth filtration ormembrane filtration can be an effective mechanism for abnormal prionprotein removal from plasma fractions.

Any acceptable assay that detects prions may be used in the quantitationaspect of the invention. Among these assays are the ELISA, SDS-Page,Western Blot, EG & G Wallac, DELFIA, Prionics assay, Enfer ELC ELISA,CEA ELISA, Conformation-dependent assays, DELFIA, and capillaryelectrophoresis, to name a few, all of which are familiar to thosehaving skill in the art.

The inventors hereof have employed the Western Blot to detect prion fromthe filtered and eluted biological fluid samples. Western blotting is amethod of used to identify and characterize PrP^(sc). The PrP^(sc) isisolated by extraction and is differentiated by its partial resistanceto proteinase K digestion. The PrP^(RES) (PrP^(sc) resistant toproteinase digestion) is identified by the migration positions of theglycosylation forms and fragments. The sensitivity of this assay isapproximately 3 logs less sensitive than the infectivity assay. Thissensitivity issue is partially overcome by centrifuging the preparation,removing the supernatant and resuspeding the prion material in a smallervolume, resulting in a concentration of the prion material. However,instead of spinning down large volumes of biological fluids such as forexample body fluids, these inventors have shown that the prions can becaptured by treating the biological fluid containing them with anaggregation aid, and then concentrating them by filtering them through afilter and later collecting them in a small volume by elution. Thistechnique can be used on a large scale to remove prions from a productstream.

This procedure will have a major impact on the use of the Western blotto determine the presence of PrP^(sc) in a biological matrix. Thisinvention allows the TSE material to be quantitatively concentratedquickly to allow for enhanced detection. When seeking to purify abiological or food solution of PrP^(sc) this invention has the advantagein the ease in which the biological or food solution and materialfilters through the large nominal pore size of the filter.

The standard Western Blot assay to confirm the specific capture of theprions relies on the captured material first being treated withProteinase K, which digests all normal prion (PrPc) but does notmarkedly digest the abnormal prion (PrP^(sc) or PrP^(res)). The digestis run in accordance with the methods of Lee et al., J Virol Methods2000, 84:77-89, on the SDS gel and transblotted to a sheet ofnitrocellulose or PVDF (polyvinylidene fluoride) membrane. The separatedPrP^(res) bands are then visualized using 3F4 or 6H4. Typical dilutionis 1:2000 for 3F4 (stock 1 mg/ml) or 1:5000 for 6H4 (stock 2.5 mg/ml),10 mL total volume in PBS Tween 20-5% nonfat milk buffer. The antibodiesare detected with goat anti-mouse IgG-HRP conjugate (1:50,000 in thesame buffer). Bands are detected with a HRP substrate usually bechemiluminescence and visualized after exposure to x-ray film. See Leeet al., supra.

Specific PrP^(sc) monoclonal antibodies like 16A18 can specifically bindthe PrP^(sc) on magnetic beads (Dynal Tosyl activated), Dynal Biotech,Oslo, Norway), and such antibodies can be used to detect presence ofPrP^(sc) rather that Western Blot methods. Most of the antibodies inthis family can capture PrP^(sc) but detection has relied on the 3F4 or6H4 in a Western Blot format as above.

Other methods to detect PrP^(sc) include ELISA and SDS-Page and othergenerally accepted detection methods as disclosed hereinabove.

In the case where the PrP^(sc) material is captured on a filter such asfor example a sterilizing filter, which filter specifically binds prionsuch as with prion-specific antibody, Western Blot methods need not beemployed to detect the PrP^(sc). Rather, a prion-specific antibody suchas a monoclonal could be employed to detect and quantitate the prion.Such an antibody includes the generic prion antibodies 6H4 or 3F4, whichrecognize both normal (PrP^(C)) and abnormal (PrP^(sc) and PrP^(res))prions. If the membrane binds all forms of prion, the relative amount ofPrP^(sc) would be very low (for instance less than about 1% of prionpresent). Specific monoclonals for abnormal prions, such as for example16A18 or 12A5, could be used to detect PrP^(sc) in the case where themembrane binds all forms (normal and abnormal) of prions. Using suchmonoclonals it should be possible to detect PrP^(sc) if the signal couldbe amplified, if necessary, using chemiluminescence substrates orpolyHRP conjugates.

The inventive methods disclosed herein results in an increase in thedetection limit of the assay by greater than 100 fold, now approachingthe infectivity assay detection limit. Using current methods availablein the art, the infectivity assay can take months to yield results,dependent upon the species under study. These inventors have also shownthat the assay can be simplified by detecting the presence of abnormalprion on the membrane surface not requiring elution.

In the use of the inventive methods of PrP^(sc) aggregation and removalwith an immunoglobulin, and in particular in the manufacture of ananti-D immunoglobulin, specifically RhoGAM Rho(D) Immune Globulin(Human), and referring to the flowsheet of FIG. 1 and the methods ofCohn et al., J. Am. Chem. Soc., Vol. 68, pages 459-475, thefractionation proceeds from whole human plasma. The plasma is cooled toabout 1° C. and is then centrifuged to separate a cold insolubleprecipitate from a supernatant. The supernatant is further fractionatedto yield Precipitate I and Supernatant I. Precipitate I which consistsprincipally of fibrinogen is discarded. Supernatant I is furtherfractionated to yield Supernatant II+III and Precipitate II+III.Supernatant II+III, which is discarded, contains alpha and beta globulinand lipids. Precipitate II+III consists principally of beta and gammaglobulins and isoagglutinins, but also contains prothrombin,plasminogen, cholesterol and other lipids. Precipitate II+III, uponfurther fractionation yields Supernatant II+III W and Precipitate II+IIIW. The beta globulin, cholesterol and other lipids are largely removedin Supernatant II+III W which is discarded. Precipitate II+III Wconsists principally of gamma globulins, isoagglutinins, plasminogen andprothrombin and some beta globulin, cholesterol and other lipids. Uponfurther fractionation, Precipitate II+III W yields SupernatantIII+Precipitate III. Precipitate III, which is discarded, containsisoagglutinins, plasminogen and prothrombin. Supernatant III consistsprincipally of gamma globulins and minor amounts of fibrinogen andlipids. The final step of the fractionation yields Precipitate II whichis essentially pure gamma G globulin. Precipitate II prepared by theprocess of the invention is an anti-Rh gamma globulin.

In the preferred methods of the invention, the immunoglobulin startingmaterial for resuspension is the Precipitate II paste from the modifiedCohn process. Lyophilized precipitate II paste may be used if theprotein is lyophilized in the presence of excipient such as thosecontemplated by U.S. Pat. No. 6,096,872. The filtration process of theinvention to capture prions in this case has preferably already beenperformed in the fractionation of Precipitate II; with reference to theabove and to FIG. 1, the filtration of the Immunoglobulin leading to thecapture of prions is performed before Precipitate II is obtained, afterobtaining Supernatant III, or, most preferably, between Supernatant IIIand Filtered Supernatant III, as shown. Such treatment of material withthe aggregation aid methanol, and at a ratio of about 4 to about 1 MeOH:Supernatant III aggregates prion in the Supernatant III, whichaggregates can then be removed using further methods of the invention.The filtration steps allowing the prion capture of the invention mayalso be done on finished immunoglobulin product. However, treatment withaggregation aid and filtration could also be performed as the finalstage of product processing so long as the treatment and filtration atthat stage do not interfere with the biological activity or otherwisecompromise the final product.

The mode of administration of the preparations of the invention maydetermine the sites and/or cells in the organism to which thecompound(s) will be delivered. The compounds purified by the methods ofthe invention can be administered alone but will generally beadministered in admixture with a pharmaceutical carrier or diluentselected with regard to the intended route of administration andstandard pharmaceutical practice. The preparations may be injectedparenterally, for example, intra-arterially or intravenously. Thepreparations may also be delivered via oral, subcutaneous, orintramuscular routes. For parenteral administration, they can be used,for example, in the form of a sterile, aqueous solution which maycontain other solutes, for example, enough salts or glucose to make thesolution isotonic.

For the oral mode of administration, the purified compositions of theinvention can be used in the form of tablets, capsules, lozenges,powders, syrups, elixirs, aqueous solutions and suspensions and thelike. In the case of tablets, carriers which can be used includelactose, sodium citrate, and salts of phosphoric acid. Variousdisintegrants such as starch, and lubricating agents such as magnesiumstearate are commonly used in tablets. For administration in capsuleform, useful diluents are lactose and high molecular weight polyethyleneglycols. When aqueous solutions are required for oral use, certainsweetening and/or flavoring agents can be added.

The substantially pure preparations of the present invention may beadministered to a subject such as a mammal, including humans. Foradministration in the treatment of afflictions, the prescribingphysician or veterinarian will ultimately determine the appropriate dosefor a given human or animal subject, and this can be expected to varyaccording to the weight, age, and response of the individual as well asthe nature and severity of the individual's symptoms.

In the case of the substantially pure anti-D immunoglobulin of theinvention, the per-dose dosage will range from about 300 ug for RhoGAM®and about 50 ug for MICRhoGAM®, each of which are administered inaccordance with the guidelines and for the purposes discussedhereinabove and in the respective product literature. Each of theproducts mentioned above can also be multi-dosed, for a total deliveryto be determined by the treating physician.

The prion-free preparations of the invention may include biologicals,medicaments, foodstuffs and feeds, and the methods of the invention maybe used in the processing of same.

Throughout this application, various patents and papers are referenced.The disclosures thereof in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described and claimed herein.

The following examples are provided for the purposes of illustrationonly and are not to be viewed as a limitation of the scope of theinvention.

EXAMPLES Example 1 Production of Rho(D) Immune Globulin Precipitate IIUsing Aggregation Aid

This Example describes a process for the fractionation of human plasmato obtain Precipitate II to be used in the production of Rho(D) immuneglobulin.

Plasma units (anti-D) (a total of approximately 943 L) were stored at 2°C. to 80° C. for four days to allow thawing. The units were pooled in astainless steel water-jacketed tank through which water at 5-10° C.circulates. The pooled plasma was stirred for thirty (30) minutes at1-3° C. The plasma was then centrifuged in a continuous flow centrifugefeeding at a rate of 1000 mL/minute. The cold insoluble supernatant(centrifuged plasma) was collected in a stainless steel. jacketed tankand stirred until a homogeneous mixture was obtained. The batch volumeat this point was 905 L of supernatant (clarified plasma).

The pH of the entire batch of supernatant was adjusted to pH 9.45 using2.172 L of 5.0N NaOH. Methanol (71%), 160.185 L was added to the pHadjusted batch, which was at −5.3° C. pH was 9.37. The batch was allowedto stand for 13.5 hours at −5.2° C. Final volume was 1067.357L.

The batch was centrifuged in a continuous flow centrifuge feeding at arate of 1000 ml/minute at −5.8° C. Supernatant I was collected in astainless steel jacketed tank, and well mixed. Precipitate I wasdiscarded as medical waste. Supernatant I was pH adjusted by adding3.132 L of conc. sodium acetate buffer, pH 4.0 and 627.444 L of 71%methanol. The batch temperature was −5.5° C. and the pH was 6.75. Thebatch was allowed to stand 14 hours.

The batch was centrifuged in a continuous flow centrifuge feeding at arate of 1000 ml/minute at −5.5° C. Precipitate II+III was transferredinto a stainless steel pot; 40.220 KG net weight was collected; this netweight Precipitate II+III was resuspended in two volumes (L) (80.440 L)of Water for Injection, U.S.P. at +1.1° C. and stirred for 45 minutesuntil a uniform suspension was obtained. Three volumes (120.660 L of0.0187M disodium phosphate was added and stirred at 2.1° C. for 30minutes.

In a stainless steel jacketed tank, 19 volumes (764.180 L) of Water forInjection, U.S.P. was cooled to 1.0° C. Using a high capacity transferpump, the batch was slowly combined with the 19 volumes of Water forInjection, U.S.P., and was stirred for 30 minutes.

A volume of 71% methanol was adjusted to equal 15 times the weight ofthe Precipitate II+III. This methanol (603.300 L) was cooled to −14° C.and using a stainless steel Sparger device and a metering pump, themethanol was added to the batch while gradually lowering the temperatureto −5.5° C. The batch was stirred for 1 hour after completion of themethanol addition. pH was 7.23. The batch was allowed to stand for 10hours 20 minutes.

Precipitate III was formed via centrifugation of the batch in acontinuous flow centrifuge feeding at a rate of 500 mL/minute at −5.8°C. The Precipitate II+III w was transferred from the bowls into astainless steel pot; the net weight was 22.760 kg.

The Precipitate II+III w was resuspended in two volumes (L) (45.520 L)of Water for Injection, U.S.P. at +1.3° C. and stirred for 45 minutesuntil a uniform suspension was obtained. Two volumes (45.520 L) of0.175M sodium acetate was added and stirred at 1.4° C. for 30 minutes.The pH of the entire batch was adjusted by addition of 0.489 L of sodiumacetate buffer, pH 4.0 in 22.760 L Water For Injection U.S.P. (totalvolume 137.049 L) to the batch and stirred for 1 hour at 1.5° C. pH was5.38. In a stainless steel jacketed tank, 13.5 volumes (307.260 L) waterfor Injection U.S.P. was cooled to +2.5° C. with stirring. Using a highcapacity transfer pump, the batch was combined with the Water forInjection, total calculated volume was 444.309 L. NaCl (6.168 L of1.33M) was added to the 22.760 Kg of Precipitate II+III, and was stirredfor 30 minutes.

A volume of 71% methanol was adjusted to equal 8.78 times the weight ofthe Precipitate II+III w. This methanol (199.833 L) was cooled to −10.5°C. and using a stainless steel Sparger device and a metering pump, themethanol was added to the batch while gradually lowering the temperatureto −6.6° C. The batch was stirred for 1 hour after completion of themethanol addition. pH was 5.38. The batch was allowed to stand for 8hours 30 minutes at −6.3° C.

Formation of Precipitate III proceeded as follows: The batch wascentrifuged in a continuous flow centrifuge feeding at a feeding rate of500 mL/minute at −6.3° C. The Supernatant III was collected in astainless steel tank. The Precipitate III was discarded as blood waste.

The filtration of the Supernatant III proceeded as follows:

The CUNO filter 90SP housing including (4) 16 sq. ft. cartridges, wasassembled in accordance with manufacturer's instruction. Sodiumacetate-methanol buffer (320 L) was cooled to −6.5° C., and was filteredthrough the filter cartridges over 55 minutes. The sodiumacetate-Methanol Buffer wash solution was blown completely out of thefilter cartridge before proceeding. The batch was filtered using theCuno filter 90SP in accordance with good manufacturing practice andemploying manufacturer's instructions. When the entire volume ofSupernatant III was filtered, the pressure in the filter housing wasreleased. Volume of filtered Supernatant III was 622 L, and was stirredat moderate speed. NaCl (1.33M, 23.387 mL) was added to Supernatant IIIslowly and stirred for 30 minutes at 6.5° C. pH was 5.38, and adjustedto 7.10 with 4.840 L of 1.0 M sodium bicarbonate and mixing for 30minutes. Methanol (100%) equal to 0.166 times the volume of SupernatantIII (103.252 L) was added to the Supernatant III using a Sparger deviceand a metering pump and the batch stirred vigorously. PH was 7.3.

Fractionation of Precipitate II was performed as follows: The batch wascentrifuged in a continuous flow centrifuge feeding at a rate of 500mL/minute at −6.3° C. and the supernatant discarded. Dry nitrogen wasused to blow out the feed lines and dry spun for 15 minutes. ThePrecipitate II (7,420 g) was transferred from the centrifuge bowls intoa tared, stainless steel pot and stored at −22.1° C. This material wasused in the viral clearance process in accordance with the methods ofco-assigned U.S. patent to Van Holten et al., U.S. Pat. No. 6,096,872issued Aug. 1, 2000.

Example 2 Elution and Detection of Prions from Example 1

The prions collected on the Cuno depth filter used to filter theSupernatant III obtained by the methods of Example 1 hereinabove areeluted, quantitated and detected using the methods of Example 3hereinbelow.

In particular, the Cuno depth filter pad used in Example 1 is removedfrom the filter housing and placed in a petri dish with 45 ml of 1.0MNaCl (elution buffer). The petri dish is placed on a rotary shaker andswirled gently for about 20-30 minutes. The filter is removed andsimilarly washed a second time with 45 mL of 2.0M NaCl (elution buffer)for 20-30 minutes.

Western Blot analysis of PrP^(sc) on the eluate is performed on eluatefrom the 1.0M NaCl elution buffer and a second Western Blot performedseparately on the eluate from the 2.0M NaCl elution, both in accordancewith the Western Blot methods of Example 3.

Example 3 Removal and Quantitation of PrP^(sc) from ImmunoglobulinPreparation

Supernatant III (SupIII) (190 mL) containing anti-D was obtained from afull-scale (approx. 450 Liters) modified Cohn-Oncley fractionation(Ortho-Clinical Diagnostics, Raritan N.J.) (See Example 1 hereinabove).The SupIII was stored at −70° C. and thawed at 25° C. just prior to theaddition of the scrapie brain homogenate (SBH), then equilibrated at atemperature of at −5.5 to −7.5° C.

Brain Homogenate

Scrapie brain homogenate (10%) was prepared using brain from hamstersinfected with 263K hamster-adapted agent. Frozen brains (approx. 3-20 as˜0.5 grams per brain) were thawed on ice, then homogenized in ninevolumes of Tris buffered saline, pH 8.0. The homogenate was clarified bycentrifugation at 1200 r.c.f. at 2-8° C. for 20 minutes. One percent(1%) lysolecithin was added to the supernatant to a final concentrationof 0.1% (w/v). This material was stored at −70° C. until use. Prior touse, the SBH was thawed in a room temperature water bath, then cuphorn-sonicated (Misonix Sonicator XL2020 with cup horn, (Heat Systems,Farmingdale, N.Y.) for approximately two minutes per milliliter untilthe solution turned from turbid to translucent. The treated homogenatewas then serially filtered through Millex® 25 mm PVDF syringe-drivenfilter units, (Millipore Corporation) 0.45/0.22/0.1 micron filters,which further clarified the material. The Supernatant III (SupIII) (200mL) from the Cohn fractionation process was spiked with the filtered SBH(1:51 dilution). The second run was filtered through a 0.22 micronfilter just prior to the start of the depth filtration to remove anyaggregates that may have formed in the mixture (see Example 5). Samplesof SHB were sampled for Western blot evaluation prior to treatment andafter sonication and filtration.

Filtration

A 47 mm CUNO Zeta Plus 90SP filter pad (Cuno Corporation, Meridan Conn.)was placed in its stainless steel filter housing. A peristaltic pump wasused to control the flow rate of the filtration to a rate of about 1ml/min. The entire filter housing was placed in an insulated sodiumchloride ice bath to cool the filter to approx. −5.5 to −7.5° C. Sodiumacetate-methanol buffer (80 ml of 0.01N sodium acetate methanol buffer,22.7% MeOH at −5.5 to −7.5° C. was used to wash the filter. TheSBH-spiked SupIII (180 ml) at −5.5 to −7.5° C. was filtered through theCUNO filter at a flow rate of 1.0 mL/minute. Aliquots of filtrate werecollected at the beginning (75 ml), middle (75 ml) and end (30 ml) ofthe filtration. The pressure of the system was monitored during theentire filtration and was about 2 psi.

Elution of PrP^(sc) from Filter

After filtration, the filter pad was removed from the filter housing andplaced, rough side up, into a beaker and washed with 45 mL of 1.0M NaClelution buffer for 20-30 minutes by gently swirling on a rotary shaker.The filter was removed and washed a second time with 45 mL of 2.0M NaClfor 20-30 minutes with gentle swirling on the rotary shaker. It would bepossible to further concentrate the PrP^(sc) by centrifugation at100,000×g for about 1 hr. at 4 degrees C., however this was notnecessary as it was sufficiently concentrated for Western Blot analysisas shown in Table 1.

A second run was identical to the first, except that the SupIII spikedwith SBH was first pre-filtered through a Millex 0.22 micron filter.

A control run was performed, cooling the filter apparatus to 0° C. andwashing the depth filter pad with 80 mL of TBS. TBS (180 mL) spiked withfiltered SBH (1:51 dilution) was filtered under the same flow rates asabove, followed by the filter washes. See Example 4.

Western Blot analysis of PrP^(sc) on the eluate was performed on eluatefrom the 1.0M NaCl elution buffer and a second Western Blot performed onthe eluate from the 2.0M NaCl elution, both in accordance with theWestern Blot methods hereinbelow.

With reference to Table 1, data is shown wherein PrP^(sc) is present ashaving been eluted from the filter after both elutions.

Western Blot

Sample Preparation

Sample preparation and assay methodology was performed in accordancewith Lee et al., J Virol Methods 2000; 84:77-89. Samples were treated ina proteinase K digestion step that is used to differentiate the PrP^(C)from the PrP^(sc). Following the proteinase K treatment the samples werecentrifuged at 20,000 r.c.f. for 1 hour at 4° C. The pellets wereresuspended in 10 μl each of 2× sodium dodecyl sulfate (SDS) samplebuffer and heated at 100° C. for five minutes. Half-log serial dilutionswere prepared prior to loading onto gels for the detection of thePrP^(RES) by Western blot, all in accordance with Lee et al. (supra)

Assay

Samples were assayed according to the method of Lee et al., supra. Bachsample was electrophoresed on a 12% SDS-Tris-glycine polyacrylamide gelfor 60 minutes at 125 constant volts. Gels were transferred tonitrocellulose membranes for 60 minutes at 125 constant mA, then soakedin TBS and blocked for 60 minutes in 5% non-fat milk. Following transferand blocking the membrane was incubated in 3F4 monoclonal antibody.After washing, the membrane was exposed to an alkalinephosphatase-conjugated anti-mouse IgG secondary antibody. The blot wasthen soaked in CDP-Star plus NitroBlock II, and then exposed to KodakXAR-2 film. A valid test was determined by the positive controlexhibiting banding at 33 kDa mark. Two smaller less intense bands thanthe 33 kDa band are also typically observed. This triplet of bands istypical Western blot image for PrP^(RES) (Lee et al, supra.).

Results

Sonication and serial membrane filtration removed all turbidity from theSBH. The subsequent depth filtration of the SBH spiked immunoglobulinpreparation reduced the PrP^(sc) concentration in the filtrate to alevel below the limits of detection of the Western blot assay (Table 1).A significant amount of the PrP^(sc) was recovered from the filter padby elution with high salt solutions Filtration of the SBH spiked SupIIIthrough a 0.22 micron filter prior to depth filtration removed PrP^(sc)to undetectable levels. The depth filtration of the SBH spiked into thebuffer control removed little or no PrP^(sc).

In Example 3 herein, membrane filtration of the sonicated SBH wasperformed prior to depth filtration of the SBH spiked SupIII in order toinsure that the depth filter would see particles no greater than 0.1micron in size. This would present the greatest challenge to the depthfilter and would allow characterization of the mechanism of PrP^(sc)removal. The SBH was first sonicated to break up the PrP^(sc) aggregatesand facilitate the membrane filtration. Despite the sonication, it wasnecessary to serially filter the SBH through progressively smallerfilters (0.45 and 0.22 micron) to minimize clogging of the 0.1 micronfilter.

Examination of the depth filter after filtration of the SBH spikedSupIII and prior to elution with the 1.0M and 2.0M NaCl solutionsrevealed a small amount of material on the surface of the depth filter.This was believed to be a precipitate formed when the SBH was added tothe SupIII, caused by the methanol present in the SupIII. In order todetermine if this precipitate contained PrP^(sc) a second run wasperformed where the SBH spiked SupIII was first pre-filtered through a0.22 micron filter prior to depth filtration. See Example 6. Thepre-filtration removed PrP^(sc) to undetectable levels, indicating thatin the prior run the PrP^(sc) was removed by precipitation andmechanical straining, rather than by adsorption to the depth filter.TABLE 1 Determination of PrP^(sc) by Western blot assay in an ImmuneGlobulin preparation spiked with scrapie brain homogenate (SBH) TotalLog₁₀ (prion unit) Mass Log₁₀ Sample Name Reduction Balance Factor*Depth Filtration of SBH spiked SupIII Spiked Load 6.9 100%  >5.2 EarlyFiltrate <2.7 0% Middle Filtrate <2.1 0% Late Filtrate <2.1 0% SaltStrip (1M) 5.2 32%  Salt Strip (2M) 5.0 1% Depth Filtration of SBHspiked SupIII with prior 0.22 μm filtration Spiked Load 7.1 100%  >3.0Spiked Load II <4.1 0% (0.22 μm filtered)† 0% Early Filtrate <2.7 0%Middle Filtrate <2.1 0% Late Filtrate <2.0 0% Salt Strip (1M) 3.2 0%Salt Strip (2M) <3.5 0% Depth Filtration of SBH spiked TBS Spiked Load7.0 100%  0.8 Early Filtrate 6.2 16%  Middle Filtrate 6.7 50%  LateFiltrate 6.0 11%  Salt Strip (1M) 4.5 0.3%   Salt Strip (2M) 4.5 0.3%  *Log₁₀ Reduction Factor is the difference between the PrP^(RES) in theSpiked Load II compared to the Filtrate.†Spiked Load II is the SBH spiked immune globulin (i.e. Spiked Load I)that was 0.22 micron filtered to remove any potential aggregates formedwith the addition of the SBH to the IgG.“<” indicates a maximum value; no PrP^(RES) was detected in any of thefiltrate samples.

Example 4 Control

In order to determine whether depth filtration would remove PrP^(sc) inthe absence of a precipitating alcohol, a control run was performedwhere the PrP^(sc) was spiked into an aqueous buffer and then depthfiltered.

The materials and procedures of Example 3 were repeated wherein the sameconcentration and volume (3.6 ml) of SBH was spiked into 180 ml of 0.1 MTris Buffered Saline (TBS). The lack of removal of PrP^(sc) from thebuffer control indicated that the depth filter did not retain theprotein, either by mechanical means (because the PrP^(sc) had previouslypassed through a 0.1 micron filter) nor by electrokinetic adsorption.See Table 1.

These data indicate that previous reports on the effectiveness of depthfiltration to remove PrP^(sc) may be misleading. Indeed, depthfiltration does remove PrP^(sc), not by the absorptive mechanism usuallyassociated with depth filtration but by mechanical straining of theprecipitated protein. The results of this study indicate that depthfiltration alone is ineffective in removing PrP^(sc). However, when usedin conjunction with a prior precipitation step, depth filtration ormembrane filtration can be an effective mechanism for abnormal prionprotein removal from plasma fractions.

Example 5 Elution of PrP^(sc) from Filter

The filter pad used in Example 3 was removed from the filter housing andplaced in a petri dish with 45 ml of 1.0M NaCl (elution buffer). Thepetri dish was placed on a rotary shaker and swirled gently for about20-30 minutes. The filter was removed and similarly washed a second timewith 45 mL of 2.0M NaCl (elution buffer) for 20-30 minutes.

Western Blot analysis of PrP^(sc) on the eluate was performed on eluatefrom the 1.0M NaCl elution buffer and a second Western Blot performedseparately on the eluate from the 2.0M NaCl elution, both in accordancewith the Western Blot methods of Example 3. With reference to Table 1,data is shown wherein PrP^(sc) is present as having been eluted from thefilter after both elutions.

Example 6 Pre Filtration of SBH in 0.22 Micron Filter

In order to determine if the precipitate observed on the filter prior tothe depth filtration step of Example 3 contained PrP^(sc), a second runwas performed where the SBH spiked SupIII was first pre-filtered througha 0.22 micron filter prior to depth filtration. The materials andprocedures of Example 3 were repeated wherein the SBH spiked SupIII waspre-filtered through a 0.22 micron filter prior to depth filtration.With reference to Table 1, it was demonstrated that the pre-filtrationremoved PrP^(sc) to undetectable levels, indicating that in the priorrun the PrP^(sc) was removed by precipitation and mechanical straining,rather than by electrostatic interaction with the depth filter.

Example 7 Elution of PrP^(sc) from Filter

Filter pads used in Example 6 were removed from the filter housing andplaced in a petri dish with 45 ml of 1.0M NaCl elution buffer. The petridish was placed on a rotary shaker and swirled gently for about 20-30minutes. The filter was removed and similarly washed a second time with45 mL of 2.0M NaCl elution buffer for 20-30 minutes.

Western Blot analysis of PrP^(sc) on the eluate was performed on eluatefrom the 1.0M NaCl elution buffer and a second Western Blot performedseparately on the eluate from the 2.0M NaCl elution, both in accordancewith the Western Blot methods of Example 3.

With reference to Table 1, data is shown wherein PrP^(sc) is present ashaving been eluted from the filter after both elutions.

Example 8 Clearance of Prions from Blood Sample

Cow whole blood (250 ml) is centrifuged at 100×g to remove the redcells. The resulting plasma is admixed with 75 ml of 22.7% methanol toaggregate the prion material. The admixture is gently swirled for 5minutes on a rotary mixer. The admixture is passed through a 47 mm CunoZeta Plus 90S filter that was prepared as in Example 3 hereinabove. Thematerial is then eluted for Western Blot assay by washing the filter padin 5 ml of 1.0 M NaCl-15mg/mL glycine solution, Following extraction andconcentration in accordance with Lee et al., 0.5 ml of this material wasanalyzed by Western Blot in accordance with the methods of Example 3.

The above procedure results in an increase in the detection limit of theassay by greater than 100 fold, now approaching the infectivity assaydetection limit. Using current methods available in the art, theinfectivity assay can take months to yield results, dependent upon thespecies under study. These inventors have also shown that the assay canbe simplified by detecting the presence of abnormal prion on themembrane surface not requiring G17 elution.

Example 9 Clearance of Prions from Urine Sample

A human urine sample (200 ml) is sedimented for 5 minutes at 3000 rpm todiscard occasional cell debris. The urine sample is admixed with 75 mlof 22.7% methanol to aggregate the prion material. The admixture isgently swirled for 5 minutes on a rotary mixer. The admixture is passedthrough a 47 mm Cuno Zeta Plus 90S filter that was prepared as inExample 3 hereinabove. The material is then eluted for Western Blotassay by washing the filter pad in 5 ml of 1.0 M NaCl-15 mg/mL glycinesolution. Following extraction and concentration in accordance with Leeet al., 0.5 ml of this material is analyzed by Western Blot inaccordance with the methods of Example 3.

It will be understood by those skilled in the art that the foregoingdescription and examples are illustrative of practicing the presentinvention, but are in no way limiting. Variations of the detailpresented herein may be made without departing from the scope and spiritof the present invention.

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 28. A method of removingprion protein from whole blood, comprising: (a) clinically centrifugingthe blood to separate the red blood cells and platelets therefrom; (b)decanting supernatant of the centrifugation of step (a) from the redblood cells and platelets; (c) admixing the supernatant with one or moreaggregation aids; (d) filtering the admixture of step (c) through amembrane or depth filter, thereby removing the prion protein fromfiltrate; and (e) adding the red blood cells and platelets separated instep (a) back to the filtrate
 29. The method of claim 28 wherein theaggregation aids are admixed together or used in series.
 30. The methodof claim 29 wherein the one or more aggregation aids comprise organicsolvents of low dielectric constant.
 31. The method of claim 30 whereinthe organic solvents are selected from the group consisting of acetoneand water-miscible alcohols.
 32. The method of claim 30 wherein theorganic solvents are selected from the group consisting of ethanol,methanol, isopropyl, isopropanol, n-propanol, isopropyl ether, ketonesand aldehydes.
 33. The method of claim 32 herein the alcohol is ethanolor methanol at a concentration of from about 2% to about 100%.
 34. Themethod of claim 29 wherein the one or more aggregation aids are selectedfrom the group containing ammonium sulfate, caprylic acid,trichloroacetic acid (TCA), dialdehydes, heteropoly acids, lactatemonohydrate (C18H21N3O 4 H2O), and the metal ions Cu2+, Ni, Zn and Ag.35. The method of claim 28 wherein when the prion protein comprisesabnormal prion protein, the one or more aggregation aids are complexingagents.
 36. The method of claim 35 wherein the complexing agent isselected from the group containing heteropolymolybdates,heteropolytungstates, sodium phosphotungstate (NaPTA), antibodies,enzymes and peptides.
 37. The method of claim 28 wherein the filter is amembrane or depth filter.
 38. The method of claim 37 wherein the filteris a depth filter.
 39. The method of claim 38 wherein the filter has apore size providing a retention of less than about 6 μm.
 40. The methodof claim 39 wherein the filter has a pore size providing a retention ofabout 0.6 to about 1.5 microns.
 41. The method of claim 38 wherein thedepth filtration is carried out using a using a depth filter having apore size providing a retention of less than about 0.6 microns. 42.(canceled)
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 75. A method of removing prion protein from whole blood,comprising: (a) clinically centrifuging the blood to separate the redblood cells and platelets therefrom; (b) decanting supernatant of thecentrifugation of step (a) from the red blood cells and platelets; (c)admixing the supernatant with methanol; (d) filtering the admixture ofstep (c) through a depth filter having a pore size providing a retentionof about 0.6 to about 1.5 microns, thereby removing the prion proteinfrom filtrate; and (e) adding the red blood cells and plateletsseparated in step (a) back to the filtrate.